1. Field of the Invention
This invention relates generally to phospholipase enzymes, polynucleotides encoding the enzymes, methods of making and using these polynucleotides and polypeptides. In alternative embodiments, the invention provides phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes, nucleic acids encoding them, antibodies that bind specifically to them, and methods for making and using them. Industrial methods and products comprising use of these phospholipases are provided. Also provided herein are methods for hydration of non hydratable phospholipids (NHPs) within a lipid matrix. The methods enable migration of NHPs to an oil-water interface thereby allowing the NHPs to be reacted and/or removed from the lipids. In certain embodiments, provided are methods for removing NHPs, hydratable phospholipids, and lecithins (known collectively as “gums”) from vegetable oils to produce a degummed oil or fat product that can be used for food production and/or non-food applications. In certain embodiments, provided herein are methods for hydration of NHPs followed by enzymatic treatment and removal of various phospholipids and lecithins. The methods provided herein can be practiced on either crude or water-degummed oils. In certain embodiment, provided herein are methods for obtaining phospholipids from an edible oil.
2. Description of Related Art
Crude vegetable oils obtained from either pressing or solvent extraction methods are a complex mixture of triacylglycerols, phospholipids, sterols, tocopherols, free fatty acids, trace metals, and other minor compounds. It is desirable to remove the phospholipids, free fatty acids and trace metals in order to produce a quality salad oil with a bland taste, light color, and a long shelf life or oil suitable for transformation into a feedstock ready for chemical or enzymatic conversion into a biofuel (methyl- or ethyl-esters), bio-plastic (epoxidized oil), and other traditional petroleum based materials.
The removal of phospholipids generates almost all of the losses associated with the refining of vegetable oils. Most phospholipid molecules possess both a hydrophilic functional group and lipophilic fatty acid chains, they tend to be excellent natural emulsifiers. The functional group in phospholipids may be any of several of a variety of known types, a few of which are illustrated in scheme 1 below.

Phospholipids containing the functional groups -choline, -inositol and -ethanolamine have the greatest affinity for water, while the acids, acid salts (Calcium (Ca), Magnesium (Mg), and Iron (Fe)), and -ethanolamine salts (Ca, Mg, and Fe) have much lower affinities for water. Phosphatidic acid and the salts of phosphatidic acid are commonly known as “non hydratable phospholipids” or NHPs. Table 1 contains relative rates of hydration of different phospholipids as reported by Sen Gupta, A. K., Fette Seifen Anstrichmittel 88 pages 79-86 (1986). and later by Segers, J. C., et al., “Degumming—Theory and Practice” published by American Oil Chemists's Society in “Edible fats and Oils processing: basic principals and modern practices: World conference proceedings”/edited by David Erickson, (1990) pages 88-93.
TABLE 1Relative Rates of HydrationRelative RatePhospholipidsof HydrationPhosphatidyl Choline (PC)100Phosphatidyl Inositol (PI)44Calcium Salt of Phosphatidyl Inositol24Phosphatidyl Ethanolamine (PE)16Phosphatidic Acid (PA)8.5Calcium Salt of Phosphatidyl Ethanolamine0.9Calcium Salt of Phosphatidic Acid0.6
Calcium, magnesium, and iron salts of phospholipids are formed by an enzyme present in oilseeds, phospholipase D (PLD). The enzyme remains dormant within the mature seed until the protective coating of the seed has been damaged during storage or seed “preparations” prior to removal of the oil. The reaction of PLD within the seed will cleave the -choline, -inositol, -serine or -ethanolamine from the phosphate group yielding the Phosphatidic Acid (PA). Additionally, since the cleavage occurs in the presence of an abundance of divalent metals (Ca, Mg, and Fe), the NHPs are formed. The phosphatidic acid calcium ion complex is shown below:

Phospholipids are commonly measured in oil as “phosphorus content” in parts per million. Table 2 sets forth the typical amounts of phospholipids present in the major oilseed crops, and the distribution of the various functional groups as a percentage of the phospholipids present in the oil.
TABLE 2Typical levels and phospholipid distributions for common oilseedsSoy OilCanola OilSunflower OilPhosphorus (ppm)400-1500200-900300-700PC (%)12-46 25-4029-52PE (%)8-3415-2517-26PA (%)2-2110-2015-30PS (%)<0.5<0.5<0.5PI (%)2-15 2-2511-22
Table 3 below provides typical phospholipid amounts and distributions for soybean gums. In Table 3, “as is” means the typical phospholipid composition removed from vegetable oil with the entrained oil (2 molecules of phospholipids and 1 molecule of oil), yielding an Acetone Insoluble content of 67%. “Normalized” means the phospholipid composition without any oil present, yielding an Acetone Insoluble content of 100%.
TABLE 3Typical phospholipid amounts and distributions for soybean gumsPercentagePercentage“As-Is”“Normalized”Phosphatidyl Choline (PC)33.947.2Phosphatidyl Ethanolamine (PE)14.319.9Phosphatidyl Serine (PS)0.40.6Phosphatidic Acid (PA)6.48.9Phosphatidyl Inositol (PI)16.823.4Total71.8100.0
The conversion of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid into either their lyso- or phospho-forms greatly changes the economics of the degumming in a modern industrial refining operation. The conversion of all of the phospholipids into their lyso-forms eliminating the neutral oil loss represents an increase in oil yield of up to 1.4%, while converting all the phospholipids into their phospho-forms represents an oil yield increase up to 3.0% for a crude oil over water degumming containing 1000 ppm of phosphorus.
Phospholipids can be partially or totally removed from vegetable oils through several different known means. The most commonly used processes in the industry are water degumming, acid degumming, caustic refining and enzymatic degumming Exemplary processes are described in U.S. Pat. Nos. 4,049,686; 4,698,185; 5,239,096; 5,264,367; 5,286,886; 5,532,163; 6,001,640; 6,103,505; U.S. Pat. Nos. 6,127,137; 6,143,545; 6,172,248; 6,548,633; 7,494,676; and 7,226,771, and U.S. publication nos. 2007/0134777, 2005/0059130, 2008/0182322, and 2009/0069587.
The existing methods are not sufficient to remove or react non-hydratable phospholipids present in the oil because the NHPs are not available to be hydrated or reacted to enable their removal.
There is a need for cost effective and efficient methods for removing NHPs, hydratable phospholipids, and lecithins (known collectively as “gums”) from vegetable oils to produce a degummed oil or fat product that can be used for food production and/or non-food applications.
Phospholipases are enzymes that hydrolyze the ester bonds of phospholipids. Corresponding to their importance in the metabolism of phospholipids, these enzymes are widespread among prokaryotes and eukaryotes. The phospholipases affect the metabolism, construction and reorganization of biological membranes and are involved in signal cascades. Several types of phospholipases are known which differ in their specificity according to the position of the bond attacked in the phospholipid molecule.
Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes are a family of eukaryotic intracellular enzymes that play an important role in signal transduction processes. The PI-PLC catalyzed reaction is:1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate(also called PIP2, phosphatidylinositol bisphosphate)+H2O1D-myo-inositol 1,4,5-trisphosphate(also called IP3, inositol triphosphate)+diacylglycerol
Families of phospholipase C (PLC) enzymes have been identified in bacteria and in eukaryotic trypanosomes. PLC enzymes belong to the family of hydrolases and phosphodiesterases. PLC participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and lipid signaling pathways in a calcium-dependent manner. PLC isoforms can differ in their mode of activation, expression levels, catalytic regulation, cellular localization, membrane binding avidity and tissue distribution. All are capable of catalyzing the hydrolysis of PIP2 into two important second messenger molecules, which go on to alter cell responses such as proliferation, differentiation, apoptosis, cytoskeleton remodeling, vesicular trafficking, ion channel conductance, endocrine function and neurotransmission. PLCs are described in, for example, Carmen, G., J. Biol. Chem. 270 (1995) 18711-18714, Jianag, Y., J. Biol. Chem., 271 (1996) 29528-29532, Waggoner, D., J. Biol. Chem. 270 (1995) 19422-19429, Molecular Probes Product Sheet 2001, and Sano et al., Am. J. Physiol. Lung Cell Mol. Physiol. 281:844-851, 2001.
Phospholipase A1 (PLA1) enzymes remove the 1-position fatty acid to produce free fatty acid and 1-lyso-2-acylphospholipid. Phospholipase A2 (PLA2) enzymes remove the 2-position fatty acid to produce free fatty acid and 1-acyl-2-lysophospholipid. PLA1 and PLA2 enzymes can be intra- or extra-cellular, membrane-bound or soluble. Intracellular PLA2 is found in almost every mammalian cell. Phospholipase C (PLC) enzymes remove the phosphate moiety to produce 1,2 diacylglycerol and a phosphate ester. Phospholipase D (PLD) enzymes produce 1,2-diacylglycerophosphate and base group.